Biological fuel cell and methods

ABSTRACT

A fuel cell has an anode and a cathode with anode enzyme disposed on the anode and cathode enzyme is disposed on the cathode. The anode is configured and arranged to electrooxidize an anode reductant in the presence of the anode enzyme. Likewise, the cathode is configured and arranged to electroreduce a cathode oxidant in the presence of the cathode enzyme. In addition, anode redox hydrogel may be disposed on the anode to transduce a current between the anode and the anode enzyme and cathode redox hydrogel may be disposed on the cathode to transduce a current between the cathode and the cathode enzyme.

This application is a continuation of application Ser. No. 13/567,903 ,filed on Aug. 6, 2012, now U.S. Pat. No. 8,435,682, which is acontinuation of application Ser. No. 12/944,551, filed on Nov. 11, 2010,now U.S. Pat. No. 8,241,797, which is a continuation of application Ser.No. 12/876,816, filed on Sep. 7, 2010, now U.S. Pat. No. 7,998,624,which is a continuation of application Ser. No. 11/768,560, filed onJun. 26, 2007, now U.S. Pat. No. 7,811,689, which is a continuation ofapplication Ser. No. 11/277,696, filed on Mar. 28, 2006, now U.S. Pat.No. 7,238,442, which is a divisional of application Ser. No. 10/385,069,filed on Mar. 10, 2003, now U.S. Pat. No. 7,018,735, which is acontinuation of application Ser. No. 09/961,621, filed on Sep. 24, 2001,now U.S. Pat. No. 6,531,239, which is a continuation of application Ser.No. 09/203,227, filed on Nov. 30, 1998, now U.S. Pat. No. 6,294,281,which claims priority to provisional application No. 60/089,900, filedon Jun. 17, 1998.

FIELD OF THE INVENTION

The present invention is, in general, directed to fuel cells and methodsof their manufacture and use. More particularly, the present inventionrelates to fuel cells capable of operation by electrolyzing compounds ina biological system and methods of their manufacture and use.

BACKGROUND OF THE INVENTION

There is interest in a variety of techniques for providing intermittentor continuous electrical power from a power source that utilizesconstituents of the environment. In the context of devices implanted ina human or animal, there is a desire to find an energy source thatutilizes the body's own chemicals for providing electrical power to thedevice. This typically includes a mechanism for converting energy storedin chemical compounds in the body to electrical energy. Such deviceshave been difficult to prepare and implement.

In outdoor situations, solar energy, wind energy, and mechanicalvibrations have been used to provide power from the environment.However, because of the diffuse nature of these sources of energy,devices with relatively large footprints are needed to provide thedesired energy. Furthermore, these sources of energy are oftenintermittent and may not be available in all situations. Anotherpotential source of energy is available from chemical energy stored inplants or their residue.

Electrochemical fuel cells have been developed to convert energy storedin chemical compounds to electrical energy. After nearly 50 years ofresearch and development, however, only the hydrogen anode/oxygencathode fuel cell operates at ambient temperatures. Fuel cells thatoperate using organic compounds have not been developed, at least inpart, because the surfaces of electrocatalysts for the oxidation oforganic compounds have not been stabilized. Fouling by intermediateoxidation products, that are strongly bound to the active sites of thecatalysts, causes loss of electrocatalyst activity. Thus, there is aneed for the development of electrochemical fuel cells that haveelectrocatalysts that are resistant to fouling and that can operateusing compounds found in biological systems.

SUMMARY OF THE INVENTION

Generally, the present invention relates to fuel cells that operateusing fuels from biological systems. One embodiment is a fuel cellhaving an anode and a cathode.

Anode enzyme is disposed on the anode and the anode is configured andarranged to electrooxidize an anode reductant in the presence of theanode enzyme. Likewise, cathode enzyme is disposed on the cathode andthe cathode is configured and arranged to electroreduce a cathodeoxidant in the presence of the cathode enzyme. In addition, anode redoxhydrogel may be disposed on the anode to transduce a current between theanode and the anode enzyme and cathode redox hydrogel may be disposed onthe cathode to transduce a current between the cathode and the cathodeenzyme.

Electrical energy is produced in the fuel cells of the present inventionas a biological fluid containing the anode reductant, such as, forexample, sugars, alcohols, carboxylic acids, carbohydrates, starches,and cellulose, and the cathode oxidant, such as, for example, O₂, flowsthrough the cell. The electrical energy produced by the fuel cell can bestored or used to power an attached device.

Another embodiment of the invention is a method of generating electricalpower in a biological system by inserting an anode and a cathode intothe biological system. A biochemical anode reductant is electrooxidizedon the anode in the presence of an anode enzyme. And a cathode oxidantis electrodreduced on the cathode, spaced apart from the anode, in thepresence of a cathode enzyme.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The Figures and the detailed description which follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, in which:

FIG. 1 is a not-to-scale cross-sectional view of one embodiment of afuel cell, according to the invention;

FIG. 2 is a perspective view of a second embodiment of a fuel cell,according to the invention;

FIG. 3 is a perspective view of a third embodiment of a fuel cell,according to the invention;

FIG. 4 is a perspective view of a fourth embodiment of a fuel cell,according to the invention; and

FIG. 5 is a perspective view of a fifth embodiment of a fuel cell,according to the invention.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is believed to be applicable to fuel cells andmethods of manufacture and use. In particular, the present invention isdirected to fuels cells capable of using compounds from biologicalsystems as fuel and methods of manufacture and use. For example, fuelcells can be made that oxidize biochemicals available in the body of ananimal, in a plant, or in plant residue. Examples of oxidizablebiochemicals include sugars, alcohols, carboxylic acids, carbohydrates,starches, and cellulose. The fuel cell can be implanted in a portion ofthe animal or plant where a fluid, such as blood or sap, flows or thefuel cell can operate utilizing tissue or fibers, particularly,cellulose, as a fuel. While the present invention is not so limited, anappreciation of various aspects of the invention will be gained througha discussion of the examples provided below.

Fuel Cell

The fuel cells of the invention typically operate using, as fuel,compounds available in a biological system. Fuel cells of the inventioncan be operated in a variety of biological systems. For example, a fuelcell may be configured for implantation into a person or animal tooperate an electrical device, such as a pacemaker, a nerve growthstimulator, a nerve stimulator for relief of chronic pain, a stimulatorfor regrowth of bone or other tissue, a drug-release valve ormicrovalve, or a fluid-flow control valve, such as a valve in a duct orin the urinary tract. Another example of a fuel cell for use with abiological system is a fuel cell that provides electricity from a plant,tree, plant residue, or the like. Typically, the fuel cells operate as abiological fluid, such as, for example, blood or sap, flows through thefuel cell. This provides a replenishing source of reactants for the fuelcell.

The fuel for the operation of the fuel cell may be provided by compoundsin blood, sap, and other biological fluids or solids. Such compounds mayinclude, for example, sugars, alcohols, carboxylic acids, carbohydrates,starches, cellulose, and dissolved or complexed oxygen (e.g., oxygencomplexed with a biomolecule such as hemoglobin or myoglobin). Examplesof compounds for electroreduction or electrooxidation in the operationof a fuel cell in an animal include glucose or lactate at the anode andoxygen, dissolved as molecular oxygen or bound in hemoglobin ormyoglobin, at the cathode.

A fuel cell 100 of the invention for use in a biological system includesan anode 102 and a cathode 104, as illustrated in FIG. 1. The anode 102and cathode 104 are separated to avoid shorting. Optionally, separationbetween the anode 102 and cathode 104 is accomplished using one or morespacers 103. The spacers 103 are often permeable, porous, microporous,and/or fibrous. Alternatively, the spacers 103 may have gaps to allowfluid to flow through the spacers 103. In some embodiments, the spacers103 may be ion selective membranes. Suitable materials for the spacers103 include, for example, polyamides (e.g., nylon), polyesters (e.g.,Dacron™), a cation exchange membrane (e.g., Nafion™), an anion exchangemembrane, porous polyolefins, polyimides, polyethers, and polyurethanes.

An anode electrolysis layer 106 is formed on at least a portion of theanode 102.

The anode electrolysis layer 106 typically includes an anode redoxpolymer and an anode enzyme. Likewise, the cathode 104 has a cathodeelectrolysis layer 108, typically including a cathode redox polymer anda cathode enzyme, formed on at least a portion of the cathode 104. Morethan one redox polymer and/or more than one enzyme can be used in eachelectrolysis layer. In some embodiments, the anode electrolysis layer106 and/or cathode electrolysis layer 108 are covered by a non-foulingcoating 109.

The enzyme in each electrolysis layer typically catalyzes anelectrochemical reaction of a cathode oxidant or anode reductant.Usually, the anode reductant is electrooxidized at the anode 102 and thecathode oxidant is electroreduced at the cathode 104. The redox polymertransduces a current between the cathode oxidant or anode reductant andthe respective electrode. In general, the cathode oxidant and anodereductant are provided within the biological system. In one embodiment,the cathode oxidant is oxygen and the anode reductant includes sugars,alcohols, and/or carboxylic acids. The fuel cell optionally includesenzymes that break down more complex molecules, such as, for example,starches and cellulose, into simpler components, such as sugars,alcohols, and/or carboxylic acids.

The physical dimensions, as well as the operational parameters, such asthe output power and voltage, are, at least in part, a function of thecomponents of the fuel cell. The open circuit voltage of the fuel cellcan range from, for example, 0.5 volts to 1.2 volts, however, the fuelcells of the invention can also produce larger or smaller voltages. Thevoltage at the maximum power point can range from, for example, 0.4 to0.8 volts. In addition, two or more fuel cells may be combined in seriesand/or in parallel to form a composite fuel cell with a larger voltageand/or current. The volumetric output power density of the fuel cell canrange from, for example, about 0.5 mW/cm³ to about 5 W/cm³, however,fuel cells can also be formed with higher or lower volumetric outputpower density. The gravimetric output power density can range from, forexample, about 5 mW/g to about 50 W/g, however, fuel cells can also beformed with higher or lower gravimetric output power density. The outputpower density depends on the flow of fluid through the fuel cell.Generally, increasing the rate of flow increases the output powerdensity.

Electrodes

The anode 102 and cathode 104 can have a variety of forms and be madefrom a variety of materials. For example, the anode and/or cathode canbe formed as plates (as shown in FIG. 1), mesh (as shown in FIG. 2),tubes (as shown in FIG. 3), or other shapes of conductive material. Theanode and/or cathode can also be a conductive film formed over an inertnon-conducting base material formed in the shape of, for example, aplate, tube, or mesh. The conductive films can be formed on thenon-conducting base material by a variety of methods, including, forexample, sputtering, physical vapor deposition, plasma deposition,chemical vapor deposition, screen printing, and other coating methods.

The anode 102 and cathode 104 are formed using a conductive material,such as, for example, metal, carbon, conductive polymer, or metalliccompound. Suitable conductive materials are typically non-corroding andcan include, for example, gold, vitreous carbon, graphite, platinum,ruthenium dioxide, and palladium, as well as other materials known tothose skilled in the art. Suitable non-conducting base materials for usewith a conductive film include plastic and polymeric materials, such as,for example, polyethylene, polypropylene, polyurethanes, and polyesters.It will be understood that the anode 102 and cathode 104 of anyparticular embodiment are not necessarily made using the same materials.

The conductive material and/or the optional non-conducting base materialare often porous or microporous. For example, the conductive materialand/or the optional non-conducting base material may be formed, forexample, as a mesh, a reticulated structure (e.g., reticulatedgraphite), a microporous film, or a film that is permeable to the anodereductant and/or cathode oxidant. The surface area of the electrode canalso be increased by roughening. Preferably, the actual exposed surfacearea of the anode and/or cathode is larger than the macroscopicgeometric surface area because the anode and/or cathode are reticulated,mesh, roughened, porous, microporous, and/or fibrous. In addition, theconductive material and/or the optional non-conducting base material canbe an ion selective membrane.

FIGS. 1 to 5 illustrate a few of the possible configurations for theanode 102 and cathode 104. The anode 102 and cathode 104 can be formedas plates and separated by optional non-conducting spacers 103, asillustrated in FIG. 1. The plates can be, for example, permeable ornon-permeable plates of conductive material that are optionally formedon a base material. The fuel cell 100 may be configured, for example, sothat biological fluid flows through and/or between the spacers 103 andthen between the anode 102 and the cathode 104, or the fuel cell 100 maybe configured so that biological fluid flows through a permeable anode102 and permeable cathode 104.

Another embodiment of a fuel cell 200 includes an anode 202 and acathode 204 formed out of a woven or mesh material, as shown in FIG. 2.The anode 202 and cathode 204 can be separated by a woven or meshnon-conducting spacer 203.

Yet another embodiment of a fuel cell 300 includes an anode 302 and acathode 304 formed as tubes and separated by an optional non-conductingspacer 303, as shown in FIG. 3. The anode 302 and cathode 304 of thisfuel cell 300 are formed, for example, using a permeable or meshmaterial to allow flow of a biological fluid through the anode 302and/or cathode 304. As an alternative, the fuel cell 300 may beconfigured for fluid flow between or through the spacers 303 and betweenthe anode 302 and cathode 304. In addition, instead of individual tubes,the anode 302 and cathode 304 may form spirals.

Another embodiment of a fuel cell 400 includes a tubular anode 402 withone or more planar cathode plates 404 in the center, as shown in FIG. 4.The tubular anode 402 and planar cathode plates 404 may be separated byan optional tubular spacer 403. Again, the fuel cell 400 may beconfigured, for example, so that biological fluid flows through apermeable anode and cathode or so that biological fluid flows betweenthe anode and/or cathode. One alternative embodiment has a tubularcathode with one or more intersecting planar anode plates at the center.

Another embodiment of a fuel cell 500 includes a tubular anode 502 witha planar cathode plate 504 at the center and a wider planar spacer 503intersecting the cathode plate 504 and positioned within the tubularanode 502 to keep the cathode plate 504 and tubular anode 502 spacedapart, as shown in FIG. 5. Again, the fuel cell 500 may be configured,for example, so that biological fluid flows through a permeable anodeand/or cathode or so that biological fluid flows between the anode andcathode. One alternative embodiment has a tubular cathode with a planaranode plate at the center.

Redox Polymers

Water, which is typically the primary mass transporting medium in manybiological systems, is an electrical insulator. Although the solubilityof many compounds is high in water, these compounds can not beelectrolyzed in the absence of transport of electrons through theaqueous medium. This can be accomplished using a redox polymer, andparticularly a redox hydrogel. Redox polymers generally provide foradequate transport of electrons if the redox polymer includes activeredox functional groups that are mobile and can carry electrons betweenthe analyte and the electrode. For example, a redox hydrogel typicallycontains a large amount of water. Water soluble reactants and productsoften permeate through the redox hydrogel nearly as fast as they diffusethrough water. Electron conduction in the redox hydrogel is throughelectron exchange between polymer segments that are mobile after thepolymer is hydrated.

The anode redox polymer and cathode redox polymer are deposited on theanode 104 and cathode 102, respectively. In general, the redox polymersinclude electroreducible and electrooxidizable ions, functionalities,species, or molecules having redox potentials. Preferably, these redoxpotentials are well-defined. The redox potentials of the redox hydrogelsare typically within a range at which water is neither electrooxidizedor electroreduced. At neutral pH and 25° C., this range is from about(−)0.65 V to about (+) 0.58 V versus the standard calomel electrode(SCE) (i.e., from about (−)0.42 V to about (+)0.81 V versus the standardhydrogen electrode (SHE)). The preferred range of the redox potentialfor the anode redox polymer is from about −0.65 V to about +0.05 V(SCE). The preferred range of the redox potential for the cathode redoxpolymer is from about +0.3 V to about +0.7 V (SCE).

The preferred redox polymers include a redox species bound to a polymerwhich can in turn be immobilized on the working electrode. In general,redox polymers suitable for use in the invention have structures orcharges that prevent or substantially reduce the diffusional loss of theredox species during the period of time that the sample is beinganalyzed. The bond between the redox species and the polymer may becovalent, coordinative, or ionic. Examples of useful redox polymers andmethods for producing them are described in U.S. Pat. Nos. 5,262,035;5,262,305; 5,320,725; 5,264,104; 5,264,105; 5,356,786; 5,593,852; and5,665,222, incorporated herein by reference. Although any organic ororganometallic redox species can be bound to a polymer and used as aredox polymer, the preferred redox species is a transition metalcompound or complex. The preferred transition metal compounds orcomplexes include osmium, ruthenium, iron, and cobalt compounds orcomplexes. In the preferred complexes, the transition metal iscoordinatively bound to one or more ligands and covalently bound to atleast one other ligand. The ligands are often mono-, di-, tri-, ortetradentate. The most preferred ligands are heterocyclic nitrogencompounds, such as, for example, pyridine and/or imidazole derivatives.For example, the multidentate ligands typically include multiplepyridine and/or imidazole rings. Alternatively, polymer-boundmetallocene derivatives, such as, for example, ferrocene, can be used.An example of this type of redox polymer is poly(vinylferrocene) or aderivative of poly(vinylferrocene) functionalized to increase swellingof the redox polymer in water.

Another type of redox polymer contains an ionically-bound redox species.Typically, this type of mediator includes a charged polymer coupled toan oppositely charged redox species. Examples of this type of redoxpolymer include a negatively charged polymer such as Nafion® (DuPont)coupled to multiple positively charged redox species such as an osmiumor ruthenium polypyridyl cations. Another example of an ionically-boundmediator is a positively charged polymer such as quaternizedpoly(4-vinyl pyridine) or poly(1-vinyl imidazole) coupled to anegatively charged redox species such as ferricyanide or ferrocyanide.The preferred ionically-bound redox species is a multiply charged, oftenpolyanionic, redox species bound within an oppositely charged polymer.

In another embodiment of the invention, suitable redox polymers includea redox species coordinatively bound to a polymer. For example, themediator may be formed by coordination of an osmium, ruthenium, orcobalt 2,2′-bipyridyl complex to poly(1-vinyl imidazole) or poly(4-vinylpyridine) or by co-polymerization of, for example, a4-vinyl-2,2′-bipyridyl osmium, ruthenium, or cobalt complex with 1-vinylimidazole or 4-vinylpyridine.

Examples of other redox species include stable quinones and species thatin their oxidized state have quinoid structures, such as Nile blue andindophenol. A preferred tetrasubstituted quinone usually has carbonatoms in the positions neighboring the oxygen-containing carbon.

The preferred redox species are osmium or ruthenium transition metalcomplexes with one or more ligands, each ligand having one or morenitrogen-containing heterocycles. Examples of such ligands includepyridine, imidazole rings, and ligands that include two or more pyridineand/or imidazole rings such as, for example, 2,2′-bipyridine;2,2′,2″-terpyridine; 1,10-phenanthroline; ligands having the followingstructures:

and derivatives thereof.

Suitable derivatives of these ligands include, for example, the additionof alkyl, alkoxy, vinyl, allyl, vinylester, and acetamide functionalgroups to any of the available sites on the heterocyclic ring,including, for example, on the 4-position (i.e., para to nitrogen) ofthe pyridine rings or on one of the nitrogen atoms of the imidazole.Typically, the alkyl, alkoxy, vinyl, and acetamide functional groups areC1 to C6 and, preferably, C1 to C3 functional groups (referring to thenumber of carbon atoms in the functional group). Suitable derivatives of2,2′-bipyridine for complexation with the osmium cation include, forexample, mono-, di-, and polyalkyl-2,2′-bipyridines, such as4,4′-dimethyl-2,2′-bipyridine, and mono-, di-, andpolyalkoxy-2,2′-bipyridines, such as 4,4′-dimethoxy-2,2′-bipyridine.Suitable derivatives of 1,10-phenanthroline for complexation with theosmium cation include, for example, mono-, di-, andpolyalkyl-1,10-phenanthrolines, such as4,7-dimethyl-1,10-phenanthroline, and mono, di-, andpolyalkoxy-1,10-phenanthrolines, such as4,7-dimethoxy-1,10-phenanthroline. Suitable derivatives for2,2′,2″-terpyridine include, for example, mono-, di-, tri-, andpolyalkyl-2,2′,2″-terpyridines, such as4,4′,4″-trimethyl-2,2′,2″-terpyridine, and mono-, di-, tri-, andpolyalkoxy-2,2′,2″-terpyridines, such as4,4′,4″-trimethoxy-2,2′,2″-terpyridine. Typically, the alkyl and alkoxygroups are C1 to C6 alkyl or alkoxy, and, preferably, C1 to C3 alkyl oralkoxy.

Suitable redox species include, for example, osmium cations complexedwith (a) two bidentate ligands, such as 2,2′-bipyridine,1,10-phenanthroline, or derivatives thereof (the two ligands notnecessarily being the same), (b) one tridentate ligand, such as2,2′,2″-terpyridine and 2,6-di(imidazol-2-yl)-pyridine, or (c) onebidentate ligand and one tridentate ligand. Suitable osmium transitionmetal complexes include, for example, [(bpy)₂OsCl]^(+/2+),[(dimet)₂OsCl]^(+/2+), [(dmo)₂OsCl]^(+/2+), [terOsCl₂]^(0/+),[trimetOsCl₂]^(0/+), and [(ter)(bpy)Os]^(2+/3+) where bpy is2,2′-bypyridine, dimet is 4,4′-dimethyl-2,2′-bipyridine, dmo is4,4′-dimethoxy-2,2′-bipyridine, ter is 2,2′,2″-terpyridine, and trimetis 4,4′,4″-trimethyl-2,2′,2″-terpyridine.

The redox species often exchange electrons rapidly between each otherand the electrode so that the complex can be rapidly oxidized and/orreduced. In general, iron complexes are more oxidizing than rutheniumcomplexes, which, in turn, are more oxidizing than osmium complexes. Inaddition, the redox potential generally increases with the number ofcoordinating heterocyclic rings.

Typically, the polymers used for the redox polymers havenitrogen-containing heterocycles, such as pyridine, imidazole, orderivatives thereof for binding as ligands to the redox species.Suitable polymers for complexation with redox species, such as thetransition metal complexes, described above, include, for example,polymers and copolymers of poly(1-vinyl imidazole) (referred to as“PVI”) and poly(4-vinyl pyridine) (referred to as “PVP”), as well aspolymers and copolymer of poly(acrylic acid) or polyacrylamide that havebeen modified by the addition of pendant nitrogen-containingheterocycles, such as pyridine and imidazole. Modification ofpoly(acrylic acid) may be performed by reaction of at least a portion ofthe carboxylic acid functionalities with an aminoalkylpyridine oraminoalkylimidazole, such as 4-ethylaminopyridine, to form amides.Suitable copolymer substituents of PVI, PVP, and poly(acrylic acid)include acrylonitrile, acrylamide, acrylhydrazide, and substituted orquaternized N-vinyl imidazole. The copolymers can be random or blockcopolymers.

The transition metal complexes typically covalently or coordinativelybind with the nitrogen-containing heterocycles (e.g., imidazole and/orpyridine) of the polymer. Alternatively, the transition metal complexesmay have vinyl functional groups through which the complexes can beco-polymerized with vinylic heterocycles, amides, nitriles, carboxylicacids, sulfonic acids, or other polar vinylic compounds, particularly,for those compounds whose polymer is known to dissolve or swell inwater.

Typically, the ratio of osmium or ruthenium transition metal complex toimidazole and/or pyridine groups ranges from 1:10 to 1:1, preferablyfrom 1:2 to 1:1, and more preferably from 3:4 to 1:1. Generally, theredox potentials of the hydrogels depend, at least in part, on thepolymer with the order of redox potentials being poly(acrylicacid)<PVI<PVP.

A variety of methods may be used to immobilize a redox polymer on anelectrode surface. One method is adsorptive immobilization. This methodis particularly useful for redox polymers with relatively high molecularweights. The molecular weight of a polymer may be increased, forexample, by cross-linking. The polymer of the redox polymer may containfunctional groups, such as, for example, hydrazide, amine, alcohol,heterocyclic nitrogen, vinyl, allyl, and carboxylic acid groups, thatcan be crosslinked using a crosslinking agent. These functional groupsmay be provided on the polymer or one or more of the copolymers.Alternatively or additionally, the functional groups may be added by areaction, such as, for example, quaternization. One example is thequaternization of PVP with bromoethylamine groups.

Suitable cross-linking agents include, for example, molecules having twoor more epoxide (e.g., poly(ethylene glycol) diglycidyl ether (PEGDGE)),aldehyde, aziridine, alkyl halide, and azide functional groups orcombinations thereof. Other examples of cross-linking agents includecompounds that activate carboxylic acid or other acid functional groupsfor condensation with amines or other nitrogen compounds. Thesecross-linking agents include carbodiimides or compounds with activeN-hydroxysuccinimide or imidate functional groups. Yet other examples ofcross-linking agents are quinones (e.g., tetrachlorobenzoquinone andtetracyanoquinodimethane) and cyanuric chloride. Other cross-linkingagents may also be used. In some embodiments, an additionalcross-linking agent is not required. Further discussion and examples ofcross-linking and cross-linking agents are found in U.S. Pat. Nos.5,262,035; 5,262,305; 5,320,725; 5,264,104; 5,264,105; 5,356,786; and5,593,852, herein incorporated by reference.

In another embodiment, the redox polymer is immobilized by thefunctionalization of the electrode surface and then the chemicalbonding, often covalently, of the redox polymer to the functional groupson the electrode surface. One example of this type of immobilizationbegins with a poly(4-vinylpyridine). The polymer's pyridine rings are,in part, complexed with a reducible/oxidizable species, such as[Os(bpy)₂Cl]^(+/2+) where bpy is 2,2′-bipyridine. Part of the pyridinerings are quaternized by reaction with 2-bromoethylamine. The polymer isthen crosslinked, for example, using a diepoxide, such as poly(ethyleneglycol) diglycidyl ether.

Carbon surfaces can be modified for attachment of a redox species orpolymer, for example, by electroreduction of a diazonium salt. As anillustration, reduction of a diazonium salt formed upon diazotization ofp-aminobenzoic acid modifies a carbon surface with phenylcarboxylic acidfunctional groups. These functional groups can be activated by acarbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC). The activated functional groups are bound with aamine-functionalized redox couple, such as, for example, the quaternizedosmium-containing redox polymer described above or2-aminoethylferrocene, to form the redox couple.

Similarly, gold and other metal surfaces can be functionalized by, forexample, an amine, such as cystamine, or by a carboxylic acid, such asthioctic acid. A redox couple, such as, for example,[Os(bpy)₂(pyridine-4-carboxylate)Cl]^(0/+), is activated by1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) toform a reactive O-acylisourea which reacts with the gold-bound amine toform an amide. The carboxylic acid functional group of thioctic acid canbe activated with EDC to bind a polymer or protein amine to form anamide.

Enzymes

The enzymes of the anode and cathode electrolysis layers catalyze anelectrochemical reaction of an anode reductant or cathode oxidant,respectively. Typically, different enzymes are provided in the anode andcathode electrolysis layers. In some embodiments, more than one enzymeis provide in the anode and/or cathode electrolysis layers. A variety ofenzymes are useful including, for example, laccase and cytochrome Coxidase on the cathode for electroreduction of oxygen; peroxidases onthe cathode for electroreduction of hydrogen peroxide; oxidases anddehydrogenases on the anode for electrooxidation of glucose, lactate,and other biochemicals; pyranose oxidase on the anode forelectrooxidation of D-glucose, L-sorbose, and D-xylose; and glucoseoxidase, oligosaccharide dehydrogenase, or pyrroloquinoline quinone(PQQ) glucose dehydrogenase on the anode for electrooxidation ofglucose. These enzymes, preferably, do not include leachable co-factors,such as NAD⁺ and NADP⁺. Other enzymes may also be included, particularlyon or near the anode, to convert more complex molecules, such asstarches and cellulose, into sugars, alcohols, and/or carboxylic acids.

One category of suitable enzymes includes thermostable enzymes which aredefined herein, unless otherwise indicated, as enzymes that function forone day, and, preferably, for five days, or more at 37° C., losing 10%or less, and, preferably, 5% or less, of their activity over the periodof use. Examples of thermostable enzyme include laccase from thethermophilic fungus myceliophthora thermophilic, cytochrome Cperioxidases from thermophilic bacterium PS3 and thermus thermophilus,peroxidase from soybean, and pyranose oxidase from the white rot fungusphlebiopsis gigantea. Other commercially available thermostable enzymesinclude L-lactate dehydrogenase from bacillus, malate dehydrogenase fromthermus species (expressed in E. coli), glucose oxidase fromaspergillus, microbial pyruvate oxidase, and urate oxidase frombacillus. Thermostable enzymes that hydrolyze larger biologicalmolecules into electrooxidizable sugars include, for example, α-amylasefrom bacillus stearothermophilus, β-amylase from aspergillus,glucan-1,4-α-glucosidase from rhizopus niveus, cellulase fromaspergillus niger, endo-1-3(4)-β-glucanase from aspergillus niger,dextranase from leuconostoc mesenteroides, α-glucosidase from bacillusstearothermophilus, β-glucosidase from caldocellum saccharolyticum,β-galactosidase from aspergillus, β-fructofuranosilidase from yeast, andlactase from aspergillus oryzae.

Alternatively, the enzyme is immobilized in a non-conducting inorganicor organic polymeric matrix to increase the thermostablity of theenzyme. Discussion regarding immobilization of an enzyme in an inorganicpolymeric matrix is found in U.S. patent application Ser. No.08/798,596, now issued as U.S. Pat. No. 5,972,199, and PCT PatentApplication No. US98/02403, now published as PCT Publication WO98/35053, incorporated herein by reference. A sol-gel polymerizationprocess provides a method for the preparation of an inorganic polymericmatrix (e.g., glass) by the polymerization of suitable monomers at ornear room-temperature. Suitable monomers include, for example, alkoxidesand esters of metallic and semiconducting elements, with preferredmetallic and semiconducting elements including Si, Al, Ti, Zr, and P.The most preferred monomers include silicon and have a silicon to oxygenratio from about 1:2 to about 1:4.

For example, enzymes can be immobilized in silica polymeric matricesmade by sol-gel processes, such as the hydrolysis of tetramethoxysilaneor another polyalkoxysilane that contains one or more silicon atoms.Condensation of the resulting silanol in the presence of the enzymeresults in entrapment of the enzyme. This process has been referred toas sol-gel immobilization. Binding of enzymes in silica or otherinorganic polymeric matrices formed from sol-gels can stabilize theenzyme. Entrapment of glucose oxidase, a glycoprotein, in a silicasol-gel matrix greatly improves the stability of the enzyme, whichretains activity when heated in water to 98° C. for 10 minutes.

An enzyme stabilized by the silica sol gel matrix can be ground to afine powder and dispersed in a silicone, preferably in an elastomericsilicone, and most preferably in a water-based elastomeric siliconeprecursor. This dispersion is then applied to the cathode as a binder ofthe enzyme. The binder preferably includes material to extract and storeoxygen from the environment. Silicone is a preferred binder in thislayer due to its ability to dissolve oxygen and its oxygen permeability.Elastomeric silicones are preferred because of high oxygen solubility.

The stability of an enzyme in an inorganic polymeric matrix depends, atleast in part, on the ionic characteristics of the enzyme and those ofthe immobilizing, often inorganic, polymeric matrix. A hydrated silicagel has an isoelectric point (pI) (i.e., the pH at which the net chargeon the molecule is zero) near pH 5. Glucose oxidase, with pI=3.8,retained its activity upon sol-gel immobilization and was stabilizedwhen immobilized in the hydrated silica gel matrix so that the half-lifeof the enzyme was increased by about 200-fold at 63° C. Lactate oxidase(pI=4.6) and glycolate oxidase (pI≈9.6), on the other hand, each lost atleast 70% of their activity upon immobilization in a hydrated silica geland the stability of these two enzymes was not greatly improved.

In contrast to the loss of activity of these enzymes in hydrated silicaalone, when poly(1-vinyl imidazole) (PVI) (a weak base) orpoly(ethyleneimine) (PEI) (a stronger base) was used to form an adductin the hydrated silica gel, the half-life of lactate oxidase (pI=4.6)increased more than 100-fold at 63° C. and the enzyme was immobilizedwithout significant loss of activity. The adduct can be formed by, forexample dissolving lactate oxidase in an aqueous buffer solution inwhich poly(1-vinyl imidazole) is co-dissolved, and the lactateoxidase-poly(1-vinyl imidazole) mixture is immobilized in silica by thesol-gel process, a stable, immobilized lactate oxidase is obtained. Thestabilized lactate oxidase can be heated in water to 90° C. for 10minutes and still retain enzymatic activity. A similar adduct whichretains enzymatic activity can be formed with poly(ethyleneimine).

Formation of an adduct between glycolate oxidase (pI≈9.6) andpoly(1-vinyl imidazole) (a weak base) did not improve the stability ofthe enzyme, but the formation of an adduct between glycolate oxidase(pI≈9.6), and poly(ethylene imine) (a stronger base) increased thehalf-life of glycolate oxidase more than 100-fold at 60° C. Forming anadduct between glucose oxidase pI=3.8 and PVI or PEI did not furtherimprove the stability of the enzyme after its immobilization in hydratedsilica.

It is thought that these functionally essential, positively chargedsurface residues (e.g., arginine) of the lactate and glycolate oxidasesmay interact with negatively charged polysilicate anions of the hydratedsilica, resulting in a decrease in activity upon sol-gel immobilization.However, when the enzyme surface is enveloped by a flexible polycationbuffer (i.e., PVI and/or PEI, depending on the isoelectric point of theenzyme) then the polysilicate anions interact with the cationic buffermolecules, and not with the cationic residues of the enzyme, therebystabilizing the enzyme by encasement in the silica gel. Thus, it isthought that PVI and PEI form adducts, acting as polycationic buffersfor enzymes such as lactate oxidase. PEI also acts as a cationic bufferfor enzymes such as glycolate oxidase. It is thought that PVI is not aneffective buffer for glycolate oxidase, because glycolate oxidase is astronger base.

In general, the addition of a polycation, such as, for example,poly(1-vinyl imidazole) or poly(ethyleneimine), prior to sol-gelimmobilization stabilizes the enzyme. Preferably, the added polycationis a more basic polyelectrolyte than the enzyme. Enzymes with highisoelectric points often need more basic polyelectrolytes forstabilization. Poly(ethyleneimine) is more basic than poly(1-vinylimidazole).

Poly(1-vinyl imidazole), a polycation at pH 7, binds at this pH toenzymes such as lactate oxidase, that are polyanions at pH 7. Thus, theaddition of a particular polymer to a particular enzyme can greatlyincrease the stability the enzyme. In the case of lactate oxidase,addition of poly(ethyleneimine), also a polybasic polymer and alsomultiply protonated at pH 7, in place of poly(1-vinyl imidazole)improved stability of the enzyme, although not as much as the additionof the preferred polymer, poly(1-vinyl imidazole). The stabilized enzymecan then be used at higher temperatures and/or for longer durations thanwould be possible if the enzyme were immobilized alone in the sol-gel.

The sol gel matrix in which an enzyme is immobilized and stabilized isoften not an electron conductor. The matrix can be modified by binding,often through covalent bonds, a redox functional group to the matrix orits precursor. Examples of suitable redox functional groups include theredox species described above for use in the redox polymer, including,for example, osmium, ruthenium, and cobalt complexes having ligandsincluding one or more pyridine and/or imidazole rings. Moreover, theredox functional group preferably includes a spacer arm covalently orcoordinatively attached a metal cation of the redox functional group orone of the ligands. One end of the spacer arm is covalently linked to,for example, silicon atoms of the matrix. The other end of the spacerarm is covalently or coordinatively linked to the redox functionalgroup. The enzyme can be immobilized in such a matrix and electrons canbe exchanged between the enzyme and the electrode using the redoxfunctional group coupled to the matrix.

In some embodiments, non-corroding, electron-conducting particles aredisposed within the matrix to increase the conductivity of the matrix;particularly, for those matrices that include attached redox functionalgroups. Examples of such particles include graphite, carbon black, gold,and ruthenium dioxide. Typically, these particles have a diameter of1 μmor less and a surface area of 1 m²/g or more, preferably, 10 m²/g ormore, and, more preferably, 100 m²/g or more. Alternatively, VOCl₃ canbe hydrolyzed to form a polymeric matrix, that, when reduced, isconducting.

In other embodiments, an enzyme is immobilized and stabilized in a solgel matrix and the enzyme catalyzes a reaction of a chemical to form aproduct that is subsequently electrooxidized or electroreduced in thepresence of a second enzyme that is electrically coupled to anelectrode. For example, glucose can react in the presence of glucoseoxidase that is stabilized in a sol gel matrix to form gluconolactoneand hydrogen peroxide. The hydrogen peroxide diffuses out of the sol gelmatrix to the proximity of the cathode and is electroreduced to water bya thermostable enzyme, such as soybean peroxidase.

Anode

At a preferred anode, one or more sugars, alcohols, and/or carboxylicacids, typically found in the biological system, are electrooxidized.Preferred anode enzymes for the electrooxidation of the anode reductantinclude, for example, PQQ glucose dehydrogenase, glucose oxidase,galactose oxidase, PQQ fructose dehydrogenase, quinohemoprotein alcoholdehydrogenase, pyranose oxidase, oligosaccharide dehydrogenase, andlactate oxidase.

One embodiment of the anode is formed using a high surface area graphitefiber/carbon black electrode using polypropylene orpolytetrafluoroethylene (e.g., Teflon™) as a binder. The anode redoxpolymer and anode enzyme are then disposed on the anode.

The anode potential can be limited by the (a) redox potential of theanode enzyme, (b) the concentration of the anode reductant at the anode,and (c) the redox potential of the anode redox polymer. Reported redoxpotentials for known anode enzymes range from about −0.4 V to about −0.5V versus the standard calomel electrode (SCE). Typically, the preferredanode redox polymers have a redox potential that is at least about 0.1 Vpositive of the redox potential of the anode enzyme. Thus, the preferredanode redox polymer can have a redox potential of, for example, about−0.3 V to −0.4 V (SCE), however, the potential of the anode redoxpolymer may be higher or lower depending, at least in part, on the redoxpotential of the anode redox enzyme. Preferred anode redox polymers forthe anode include [(dmo)₂OsCl]^(+/2+), [terOsCl₂]^(0/+), and[trimetOsCl₂]^(0/+) coupled to either PVI or poly(acrylic acid) or acopolymer of 4-vinyl pyridine or 1-vinyl imidazole.

In some embodiments, one or more additional enzymes are provided inproximity to or disposed on the anode. The additional enzyme or enzymesbreak down starch, cellulose, poly- and oligosaccharides, disaccharides,and trisaccharides into the sugars, alcohols, and/or carboxylic acidsthat are used as fuel. Examples of such catalysts include α-amylase frombacillus stearothermophilus, β-amylase from aspergillus,glucan-1,4-α-glucosidase from rhizopus niveus, cellulase fromaspergillus niger, endo-1-3(4)-β-glucanase from aspergillus niger,dextranase from leuconostoc mesenteroides, α-glucosidase from bacillusstearothermophilus, β-glucosidase from caldocellum saccharolyticum,β-galactosidase from aspergillus, β-fructofuranosilidase from yeast, andlactase from aspergillus oryzae.

Cathode

In one embodiment of the fuel cell, the cathode reduces gaseous O₂ thatis typically dissolved in the biological fluid or originating from theair. In another embodiment of the fuel cell, hydrogen peroxide is formedin a non-enzyme-catalyzed electrode reaction or in an enzyme-catalyzedreaction on or off the cathode and then the hydrogen peroxide iselectroreduced at the cathode. Preferred cathode enzymes for thereduction of O₂ and H₂O₂ include, for example, tyrosinase, horseradishperoxidase, soybean peroxidase, other peroxidases, laccases, and/orcytochrome C peroxidases.

One embodiment of the cathode includes a porous membrane formed over atleast a portion of cathode. The porous membrane has an O₂ or H₂O₂permeable, hydrophobic outer surface and an O₂ or H₂O₂ permeablehydrophilic inner surface. In another embodiment, the cathode includesan outer layer of a hydrophobically modified porous silicate carboncomposite, formed of an alkyltrialkoxysilane precursor, and carbonblack. The inner layer is a hydrophilic silca-carbon composite. Inanother embodiment, the electrode is a microporous Teflon PTFE boundacetylene/carbon black electrode. The inner surface is plasma processedto make it hydrophilic. The redox polymer and enzyme are deposited onthe inner surface of the cathode. When the cathode is exposed to O₂originating in blood or a body fluid, the cathode may only includehydrophilic surfaces in contact with the O₂ transporting biologicalfluid.

The cathode potential can be limited by the (a) redox potential of thecathode enzyme, (b) the concentration of the cathode oxidant at thecathode, and (c) the redox potential of the cathode redox polymer.Reported redox potentials for known O₂ reducing enzymes range from about+0.3 V to about +0.6 V versus the standard calomel electrode (SCE).Typically, the preferred cathode redox polymer has a redox potentialthat is at least about 0.1 V negative of the redox potential of theenzyme. Thus, the preferred redox polymer has redox potential of, forexample, about +0.4 to +0.5 V (SCE), however, the potential of thecathode redox polymer may be higher or lower depending, at least inpart, on the redox potential of the cathode redox enzyme.

For osmium complexes used as the cathode redox polymer, typically, atleast four, usually, at least five, and, often, all six of the possiblecoordination sites of the central osmium atom are occupied by nitrogenatoms. Alternatively, for complexes of ruthenium used as the cathoderedox polymer, typically, four or fewer, and, usually, three or fewer ofthe possible coordination sites are nitrogen occupied. Preferred cathoderedox polymers include [(ter)(bpy)Os]^(2+/3+) coupled to PVI or PVP.

Non-fouling Coatings

An optional non-fouling coating is formed over at least that portion ofthe electrodes of the fuel cell. The non-fouling coating prevents orretards the penetration of macromolecules, such as proteins, having amolecular weight of 5000 daltons or more, into the electrodes of thefuel cell. This can be accomplished using a polymeric film or coatinghaving a pore size that is smaller than the biomolecules that are to beexcluded or having anionic and/or cationic functional groups that repelcationic or anionic macromolecules, respectively. Such biomolecules mayfoul the electrodes and/or the electrolysis layer thereby reducing theeffectiveness of the fuel cell and altering the expected electricalpower generation. The fouling of the electrodes may also decrease theeffective life of the fuel cell.

For example, the electrodes of the fuel cell may be completely orpartially coated on their exterior with a non-fouling coating. Apreferred non-fouling coating is a polymer, such as a hydrogel, thatcontains at least 20 wt. % fluid when in equilibrium with theanalyte-containing fluid. Examples of suitable polymers are described inU.S. Pat. No. 5,593,852, incorporated herein by reference, and includecrosslinked polyethylene oxides, such as polyethylene oxidetetraacrylate and diacrylate. For example, polyethylene oxide (“PEO”)chains, typically of 8-18 kilodaltons are terminally modified withreactive groups, such as acrylates and methacrylates. In addition,diesters of PEO can be reacted with star-dendrimer PEO polyamines toform the non-fouling coatings.

Implantation in a Blood Vessel

For continuously producing power, the reactant-carrying fluid typicallyflows through the fuel cell, so as to replenish the anode reductantand/or cathode oxidant exhausted by reacting at the anode and/orcathode, respectively. When the fuel cell is implanted in a blood vesselof an animal (e.g., human, mammal, bird, or fish), the fluid may bepumped through the fuel cell by the heart, obviating any need for amechanical pump and thereby reducing the weight and volume of the systemcontaining the fuel cell.

Use and Storage of Electrical Power Generated by the Fuel Cell

The electrical power generated by the fuel cell can be used to operate avariety of devices, including, for example, medical or other devicesimplanted in a human or animal. Examples of medical devices includepacemakers, nerve growth stimulators, nerve stimulators for relief ofchronic pain, stimulators for regrowth of bone or other tissue,drug-release valves or microvalves, and fluid-flow control valves, suchas a valve in a duct or in the urinary tract. The electrical power canalso be used to operate external devices connected to the fuel cell(e.g., a fuel cell implanted in a plant or tree). In addition, theelectrical power can be stored in a storage device, such as a capacitivestorage element or battery, for later use.

The present invention should not be considered limited to the particularexamples described above, but rather should be understood to cover allaspects of the invention as fairly set out in the attached claims.Various modifications, equivalent processes, as well as numerousstructures to which the present invention may be applicable will bereadily apparent to those of skill in the art to which the presentinvention is directed upon review of the instant specification.

What is claimed:
 1. An electrochemical cell for positioning in abiological system comprising oxygen, comprising: a substrate comprisinga first face and a second face; an anode disposed on the first face ofthe substrate, wherein the anode comprises an anode enzyme and amediator, wherein the enzyme and the mediator are crosslinked; and acathode disposed on the second face of the substrate.
 2. Theelectrochemical cell according to claim 1, wherein the mediator is ametallocene derivative.
 3. The electrochemical cell according to claim2, wherein the metallocene derivative is ferrocene.
 4. Theelectrochemical cell according to claim 1, wherein the mediatorcomprises a transition metal complex.
 5. The electrochemical cellaccording to claim 4, wherein the transition metal complex comprisesruthenium or osmium.
 6. The electrochemical cell according to claim 1,wherein the anode comprises a conductive material selected from thegroup consisting of gold, carbon, platinum, ruthenium dioxide andpalladium.
 7. The electrochemical cell according to claim 6, wherein theanode comprises carbon or gold.
 8. The electrochemical cell according toclaim 1, wherein the substrate surface is roughened.
 9. Theelectrochemical sensor according to claim 1, wherein the anode enzyme isa glucose-responsive enzyme.
 10. The electrochemical cell according toclaim 9, wherein the glucose-responsive enzyme is glucose oxidase orglucose dehydrogenase.
 11. The electrochemical cell according to claim9, wherein the glucose-responsive enzyme further comprises an enzymecofactor.
 12. The electrochemical cell according to claim 11, whereinthe enzyme cofactor is pyrroloquinoline quinone (PQQ).
 13. Theelectrochemical cell according to claim 1, wherein the cathode comprisesplatinum.
 14. The electrochemical cell according to claim 1, wherein thecathode further comprises a cathode enzyme.
 15. The electrochemical cellaccording to claim 14, wherein the cathode enzyme is an enzyme selectedfrom the group consisting of tyrosinase, horseradish peroxidase, soybeanperoxidase, laccase and cytochrome C peroxidase.
 16. The electrochemicalcell according to claim 15, wherein the cathode enzyme is laccase orcytochrome C peroxidase.
 17. The electrochemical cell according to claim1, wherein at least a portion of the electrochemical cell is configuredto be subcutaneously positioned in a subject.
 18. The electrochemicalcell according to claim 1, wherein the electrochemical cell furthercomprises a mass transport limiting membrane disposed over the anode andcathode.
 19. The electrochemical cell according to claim 18, wherein themass transport limiting membrane is configured to limit contact ofmacromolecules of 5000 daltons or greater with the anode and cathode.20. The electrochemical cell according to claim 18 wherein the masstransport limiting membrane is a hydrogel.
 21. The electrochemical cellaccording to claim 20, wherein the hydrogel comprises at least 20 wt %fluid when in equilibrium with analyte-containing fluid.